Regulation of endometrial repair and its impact on heavy menstrual bleeding
Date
25/11/2011Author
Maybin, Jacqueline Ann
Metadata
Abstract
INTRODUCTION: The human endometrium has a remarkable capacity for efficient
cyclical repair following the inflammatory process of menstruation. Defective postmenstrual
repair may contribute to the common complaint of heavy menstrual
bleeding (HMB). The mechanisms and factors involved in endometrial repair are still
to be fully elucidated. Endometrial function is governed by the ovarian hormones and
pre-menstrually progesterone levels decline as the corpus luteum regresses.
Consequently, the synthesis of prostaglandins (PG) is increased, namely PGE2 and
the potent vasoconstrictor PGF2α. Subsequent vasoconstriction of endometrial spiral
arterioles is believed to result in a transient hypoxic episode in the upper endometrial
layer.
Therefore, the aims of this thesis were to determine (i) the endometrial expression of
putative repair factors across the menstrual cycle (ii) the regulation of these factors
by hypoxia, PGE2 and PGF2α (ii) the role of hypoxia inducible factor (HIF)-1α in
endometrial repair and (iii) differences in endometrium from women with objectively
measured HMB (>80ml) and normal controls (<80ml).
METHODS/RESULTS:
Putative repair factors, with known angiogenic, mitogenic and
proliferative functions, were identified in human endometrial samples by quantitative
reverse transcription PCR and immunohistochemistry. Interleulin-8 (IL-8), vascular
endothelial growth factor (VEGF), adrenomedullin (AM), connective tissue growth
factor (CTGF) and endothelin-1 (ET-1) were all maximally expressed during the
menstrual and/or proliferative phases of the cycle, consistent with the onset of
endometrial repair. Endometrial cells and tissue explants treated with 100nM
PGE2/F2α and/or hypoxia (0.5% O2) revealed up-regulation of IL-8, VEGF, AM and
CTGF. An in vitro progesterone antagonism model revealed that progesterone
withdrawal, hypoxia and prostaglandins are all necessary for significant increases in
repair factor expression in endometrial tissue. HIF-1α was detected in human
endometrium but exclusively in the late-secretory and menstrual phases. Using shorthairpin
RNA against HIF-1α, it was determined that hypoxia up-regulated these
factors via HIF-1α, whereas PGF2α acted in a HIF-1α independent manner to increase repair factor expression. Finally, whole genome array analysis was performed on
menstrual endometrium from women with objectively measured heavy and normal
menstrual bleeding to provide an unbiased comparison of gene expression. 259
transcripts displayed significant changes between the two groups. Five candidate
genes were validated using Q-RT-PCR. Bioinformatic analysis of the differentially
expressed gene set identified bioprocesses that included positive regulation of
biological and cellular processes, leukocyte differentiation, regulation of apoptosis
and response to stress/hypoxia. The presence of HIF-1α protein was examined in
menstrual endometrial tissue nuclear protein extracts by Western blot, revealing
significantly decreased levels in women with HMB versus normal controls.
Furthermore, the mRNA expression of known target genes of HIF-1α (VEGF,
CXCR4) was also significantly decreased in these women. The functional impact of
endometrial HIF-1α was assessed using an in vitro angiogenic assay. Silencing of
HIF-1α in endometrial cells significantly reduced the angiogenic potential of culture
supernatants when compared to untransfected cells or cells transfected with a
scrambled sequence.
CONCLUSIONS: Repair factors are significantly increased in the human endometrium
following the onset of menstruation. Progesterone withdrawal, hypoxia via HIF-1α
and prostaglandins appear necessary for the regulation of these factors at this time.
Menstrual endometrium displays significant differences in gene expression and HIF-
1α protein levels between women with HMB and normal controls. The findings of
this thesis contribute to the existing literature on both the physiological process of
endometrial repair and the pathogenesis of HMB. Extension of this work may allow
the identification of novel therapeutic targets for the treatment of this common,
debilitating condition.